Assembly of 43 human Y chromosomes reveals extensive complexity and variation.

The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date1 and led to its systematic omission from genomic analyses. Here we present de novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes2. Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established1 boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome.

Somatic nuclear mitochondrial DNA insertions are prevalent in the human brain and accumulate over time in fibroblasts.

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in nonhuman species and recently demonstrated to occur in rare instances from one human generation to the next. Here, we investigated numtogenesis dynamics in humans in 2 ways. First, we quantified Numts in 1,187 postmortem brain and blood samples from different individuals. Compared to circulating immune cells (n = 389), postmitotic brain tissue (n = 798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples, we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex (DLPFC) compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, an increase in the number of brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment (NCI) who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures whole-genome sequencing design in a human fibroblast model that recapitulates several molecular hallmarks of aging. These longitudinal experiments revealed a gradual accumulation of 1 Numt every ~13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven by cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derived SURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7-fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human postmitotic tissues produces functionally relevant human Numts over timescales shorter than previously assumed.

The Utility of the Ultrasound "Superimposed-Line" Sign at the Junction of the Vomer and Maxilla in First-Trimester Screening for Fetal Cleft Palate: A Case-Control Study.

INTRODUCTION: The current retrospective case-control study evaluates the diagnostic value of screening for a fetal cleft palate by using the ultra-sound-based observation of the "superimposed-line" sign appearing at the junction of the vomer and maxilla in the first trimester of pregnancy. METHODS: Retrospective analyses were performed of ultrasonographic images of nuchal translucency obtained during the first trimester of pregnancy (11-13+6 weeks) from 45 fetuses with a cleft palate later confirmed following parturition or induced labor (cases) and 4,500 normal fetuses confirmed after parturition (controls). Ultrasonographic features of the "superimposed-line" sign were observed and recorded, and between-group comparisons were performed. RESULTS: The "superimposed-line" sign was absent in 39 cases (86.67%), including 4 (8.89%) with simple secondary hard palate cleft and 35 (77.78%) with secondary hard palate cleft complicated by a primary cleft palate. The "superimposed-line" sign was shown in 6 cases (13.33%), including 2 (4.44%) with a simple secondary soft palate cleft, 1 (2.22%) with a simple secondary bifid uvula, and 3 (6.67%) with a simple primary cleft palate. Among the 4,500 controls, 31 fetuses showed an absence of the "superimposed-line" sign (0.69%) and 4,469 showed the "superimposed-line" sign (99.31%). The between-group difference was significant (p < 0.05). The sensitivity, specificity, positive predictive value, and negative predictive values of the "superimposed-line" sign in the first trimester of pregnancy for predicting fetal cleft palate were 86.67% (39/45), 99.31% (4,469/4,500), 55.71% (39/70), and 99.86% (4,469/4,475), respectively. CONCLUSION: The "superimposed-line" sign did not appear in fetuses with secondary hard palate cleft and primary cleft palate only when a secondary hard palate cleft is present. The sign appeared in normal fetuses and those with a simple primary cleft palate, simple secondary soft palate cleft, or a simple secondary bifid uvula. Based on these results, we propose that the "superimposed-line" sign in the mid-sagittal plane of the fetal face in the first trimester of pregnancy (11-13+6 weeks) is an important tool in screening for fetal cleft palate, especially secondary hard palate cleft.

Identification and characterization of occult human-specific LINE-1 insertions using long-read sequencing technology.

Long Interspersed Element-1 (LINE-1) retrotransposition contributes to inter- and intra-individual genetic variation and occasionally can lead to human genetic disorders. Various strategies have been developed to identify human-specific LINE-1 (L1Hs) insertions from short-read whole genome sequencing (WGS) data; however, they have limitations in detecting insertions in complex repetitive genomic regions. Here, we developed a computational tool (PALMER) and used it to identify 203 non-reference L1Hs insertions in the NA12878 benchmark genome. Using PacBio long-read sequencing data, we identified L1Hs insertions that were absent in previous short-read studies (90/203). Approximately 81% (73/90) of the L1Hs insertions reside within endogenous LINE-1 sequences in the reference assembly and the analysis of unique breakpoint junction sequences revealed 63% (57/90) of these L1Hs insertions could be genotyped in 1000 Genomes Project sequences. Moreover, we observed that amplification biases encountered in single-cell WGS experiments led to a wide variation in L1Hs insertion detection rates between four individual NA12878 cells; under-amplification limited detection to 32% (65/203) of insertions, whereas over-amplification increased false positive calls. In sum, these data indicate that L1Hs insertions are often missed using standard short-read sequencing approaches and long-read sequencing approaches can significantly improve the detection of L1Hs insertions present in individual genomes.

Hypoxia modulation by dual-drug nanoparticles for enhanced synergistic sonodynamic and starvation therapy.

BACKGROUND: Sonodynamic therapy (SDT) is an emerging non-invasive therapeutic technique. SDT-based cancer therapy strategies are presently underway, and it may be perceived as a promising approach to improve the efficiency of anti-cancer treatment. In this work, multifunctional theranostic nanoparticles (NPs) were synthesized for synergistic starvation therapy and SDT by loading glucose oxidase (GOx, termed G) and 5,10,15,20-tetrakis (4-chlorophenyl) porphyrin) Cl (T (p-Cl) PPMnCl, termed PMnC) in Poly (lactic-co-glycolic) acid (PLGA) NPs (designated as MG@P NPs). RESULTS: On account of the peroxidase-like activity of PMnC, MG@P NPs can catalyze hydrogen peroxide (H2O2) in tumor regions to produce oxygen (O2), thus enhancing synergistic therapeutic effects by accelerating the decomposition of glucose and promoting the production of cytotoxic singlet oxygen (1O2) induced by ultrasound (US) irradiation. Furthermore, the NPs can also serve as excellent photoacoustic (PA)/magnetic resonance (MR) imaging contrast agents, effectuating imaging-guided cancer treatment. CONCLUSION: Multifunctional MG@P NPs can effectuate the synergistic amplification effect of cancer starvation therapy and SDT by hypoxia modulation, and act as contrast agents to enhance MR/PA dual-modal imaging. Consequently, MG@P NPs might be a promising nano-platform for highly efficient cancer theranostics.

Alleviation Effects and Mechanisms of Low-intensity Focused Ultrasound on Pain Triggered by Soft Tissue Injury.

OBJECTIVES: Pain caused by soft tissue injury (STI) is always intractable and will eventually result in physical and psychological problems. This experiment aimed to assess the efficacy and mechanisms of low-intensity focused ultrasound (LIFU) for pain-related STI. METHODS: Rabbits (n = 30) with STI were given fixed treatment for 20 seconds and then mobile treatment for 60 seconds daily for 10 consecutive days by an LIFU device with a power output of 5 to 6 W and a frequency of 0.8 MHz. To evaluate the degree of pain, the levels of ß-endorphin in serum were measured by an enzyme-linked immunosorbent assay before and 5 to 10 minutes after the 1st, 3rd, 7th, and 10th treatments. The pain threshold was measured by an electronic analgesy meter on the 1st, 3rd, 7th, 10th, 17th, and 24th days after the start of the treatment. To investigate inflammation, prostaglandin E2 , interleukin-1ß, and 5-hydroxytryptamine levels were detected by an enzyme-linked immunosorbent assay, and nuclear factor κB messenger RNA levels were determined by a real-time quantitative polymerase chain reaction at the same time as the pain threshold was tested. RESULTS: Compared with non-LIFU groups, ß-endorphin levels and pain thresholds were significantly increased (P < .05), whereas nuclear factor- κB messenger RNA, prostaglandin E2 , interleukin- 1ß, and 5-hydroxytryptamine levels were significantly reduced (P < .05) after LIFU treatment in rabbits with STI. CONCLUSIONS: Low-intensity focused ultrasound can alleviate pain induced by STI and could have further clinical applications.

Efficient CNV breakpoint analysis reveals unexpected structural complexity and correlation of dosage-sensitive genes with clinical severity in genomic disorders.

Genomic disorders are the clinical conditions manifested by submicroscopic genomic rearrangements including copy number variants (CNVs). The CNVs can be identified by array-based comparative genomic hybridization (aCGH), the most commonly used technology for molecular diagnostics of genomic disorders. However, clinical aCGH only informs CNVs in the probe-interrogated regions. Neither orientational information nor the resulting genomic rearrangement structure is provided, which is a key to uncovering mutational and pathogenic mechanisms underlying genomic disorders. Long-range polymerase chain reaction (PCR) is a traditional approach to obtain CNV breakpoint junction, but this method is inefficient when challenged by structural complexity such as often found at the PLP1 locus in association with Pelizaeus-Merzbacher disease (PMD). Here we introduced 'capture and single-molecule real-time sequencing' (cap-SMRT-seq) and newly developed 'asymmetry linker-mediated nested PCR walking' (ALN-walking) for CNV breakpoint sequencing in 49 subjects with PMD-associated CNVs. Remarkably, 29 (94%) of the 31 CNV breakpoint junctions unobtainable by conventional long-range PCR were resolved by cap-SMRT-seq and ALN-walking. Notably, unexpected CNV complexities, including inter-chromosomal rearrangements that cannot be resolved by aCGH, were revealed by efficient breakpoint sequencing. These sequence-based structures of PMD-associated CNVs further support the role of DNA replicative mechanisms in CNV mutagenesis, and facilitate genotype-phenotype correlation studies. Intriguingly, the lengths of gained segments by CNVs are strongly correlated with clinical severity in PMD, potentially reflecting the functional contribution of other dosage-sensitive genes besides PLP1. Our study provides new efficient experimental approaches (especially ALN-walking) for CNV breakpoint sequencing and highlights their importance in uncovering CNV mutagenesis and pathogenesis in genomic disorders.

Low-Intensity Pulsed Ultrasound Relieves Mild and Severe Myelosuppression Induced by Cyclophosphamide in Rabbits.

OBJECTIVE: This study aimed to investigate the effect of low-intensity pulsed ultrasound (LIPUS) on cyclophosphamide (CTX)-induced rabbit myelosuppression. METHODS: Rabbits (n = 90) were randomly divided into a mild myelosuppression group (n = 40), a severe myelosuppression group (n = 40), and a normal control group (group Cu28 ; n = 10). The mild and severe myelosuppression models were established by daily ear vein injection of 15- and 40-mg/kg CTX for 4 continuous days, respectively. Then they were randomly divided into LIPUS groups (Au and Bu ) and control groups (Ac and Bc ). LIPUS was applied once per day for 20 minutes for 7 (Au7 and Bu7 ) and 28 (Au28 and Bu28 ) days. Physical conditions, mortality, blood cell counts, and bone marrow proliferation were calculated. Erythropoietin interleukin 3, and granulocyte-macrophage colony-stimulating factor levels were measured by an enzyme-linked immunosorbent assay. Flow cytometry was used to detect the granulocyte phagocytosis rate. Hematoxylin-eosin staining was performed to analyze changes of skin and muscle. RESULTS: Compared with the control group, LIPUS improved the number of peripheral blood cells (P < .05) and bone marrow nucleated cells and reduced the mortality of rabbits with myelosuppression of different degrees. Long-term treatment for 28 days had no effect on the levels of erythropoietin, interleukin 3, and granulocyte-macrophage colony-stimulating factor and granulocyte phagocytosis (P > .05). The parts of the skin where LIPUS was applied did not show any burning marks, and the muscle tissue in the path of LIPUS acoustic channels showed no obvious pathologic changes. CONCLUSIONS: Low-intensity pulsed ultrasound is a safe and effective method to relieve CTX-induced myelosuppression.

knnAUC: an open-source R package for detecting nonlinear dependence between one continuous variable and one binary variable.

BACKGROUND: Testing the dependence of two variables is one of the fundamental tasks in statistics. In this work, we developed an open-source R package (knnAUC) for detecting nonlinear dependence between one continuous variable X and one binary dependent variables Y (0 or 1). RESULTS: We addressed this problem by using knnAUC (k-nearest neighbors AUC test, the R package is available at https://sourceforge.net/projects/knnauc/ ). In the knnAUC software framework, we first resampled a dataset to get the training and testing dataset according to the sample ratio (from 0 to 1), and then constructed a k-nearest neighbors algorithm classifier to get the yhat estimator (the probability of y = 1) of testy (the true label of testing dataset). Finally, we calculated the AUC (area under the curve of receiver operating characteristic) estimator and tested whether the AUC estimator is greater than 0.5. To evaluate the advantages of knnAUC compared to seven other popular methods, we performed extensive simulations to explore the relationships between eight different methods and compared the false positive rates and statistical power using both simulated and real datasets (Chronic hepatitis B datasets and kidney cancer RNA-seq datasets). CONCLUSIONS: We concluded that knnAUC is an efficient R package to test non-linear dependence between one continuous variable and one binary dependent variable especially in computational biology area.

Correlation between frequency of non-allelic homologous recombination and homology properties: evidence from homology-mediated CNV mutations in the human genome.

Non-allelic homologous recombination (NAHR) is one of the key mechanisms of DNA rearrangement. NAHR occurring between direct homologous repeats can generate genomic copy number variation (CNV) and make significant contributions to both genome evolution and human diseases such as cancer. Intriguingly, previous observations on the rare CNVs at certain genomic disorder loci suggested that NAHR frequency could be dependent on homology properties. However, such a correlation remains unclear at the other NAHR-mediated CNV loci, especially the common CNVs in human populations. Different from the rare CNVs associated with genomic disorders, it is challenging to identify de novo NAHR events at common CNV loci. Therefore, our previously proposed statistic M was employed in estimating relative mutation rate for the NAHR-mediated CNVs in human populations. By utilizing generalized regression neural network and principal component analysis in studying 4330 CNVs ascertained in 3 HapMap populations, we identified the CNVs mediated by NAHR between paired segmental duplications (SDs) and further revealed the correlations between SD properties and NAHR probability. SD length and inter-SD distance were shown to make major contributions to the occurrence of NAHR, whereas chromosomal position and sequence similarity of paired SDs are also involved in NAHR. An integrated effect of SD properties on NAHR frequency was revealed for the common CNVs in human populations. These observations can be well explained by ectopic synapsis in NAHR together with our proposed model of chromosomal compression/extension/looping (CCEL) for homology mis-pairing. Our findings showed the important roles of SDs in NAHR and human genomic evolution.

CNV instability associated with DNA replication dynamics: evidence for replicative mechanisms in CNV mutagenesis.

Copy number variation (CNV) in the human genome is of vital importance to human health and evolution of our species. However, much of the molecular basis of CNV mutagenesis remains to be elucidated. Considering the DNA replication model of 'fork stalling and template switching' for CNV formation, we hypothesized that replication fork progression could be important for CNV mutagenesis. However, molecular assays of replication fork progression at the genome level are technically challenging. Instead, we conducted an estimation of DNA replication dynamics, as the statistic R, using the readily available data of replication timing. Small R-values can reflect 'slowed' replication, which could result from less fork initiation, reduced fork speed or fork barriers. We generated genome-wide profiles of R in the genomes of human, mouse and Drosophila. Intriguingly, the CNV breakpoints in all three genomes showed significantly biased distributions toward the genomic regions with small R-values, suggesting potential replication stress-induced CNV instability. Notably, among the human CNVs with distinct breakpoint junction characteristics, the homology-mediated and VNTR-mediated CNVs contribute the most to the correlation between CNV instability and the statistic R, consistent with the recent findings in the C. elegans and yeast genomes of repeat-induced DNA replication error and consequent CNV formation. The statistic R may reflect both replication stress and the effect of local genome architecture on fork progression. Our concordant observations suggest an important role for DNA replicative mechanisms in CNV mutagenesis and genome instability.

Predictive model for inflammation grades of chronic hepatitis B: Large-scale analysis of clinical parameters and gene expressions.

BACKGROUND: Liver biopsy is the gold standard to assess pathological features (eg inflammation grades) for hepatitis B virus-infected patients although it is invasive and traumatic; meanwhile, several gene profiles of chronic hepatitis B (CHB) have been separately described in relatively small hepatitis B virus (HBV)-infected samples. We aimed to analyse correlations among inflammation grades, gene expressions and clinical parameters (serum alanine amino transaminase, aspartate amino transaminase and HBV-DNA) in large-scale CHB samples and to predict inflammation grades by using clinical parameters and/or gene expressions. METHODS: We analysed gene expressions with three clinical parameters in 122 CHB samples by an improved regression model. Principal component analysis and machine-learning methods including Random Forest, K-nearest neighbour and support vector machine were used for analysis and further diagnosis models. Six normal samples were conducted to validate the predictive model. RESULTS: Significant genes related to clinical parameters were found enriching in the immune system, interferon-stimulated, regulation of cytokine production, anti-apoptosis, and etc. A panel of these genes with clinical parameters can effectively predict binary classifications of inflammation grade (area under the ROC curve [AUC]: 0.88, 95% confidence interval [CI]: 0.77-0.93), validated by normal samples. A panel with only clinical parameters was also valuable (AUC: 0.78, 95% CI: 0.65-0.86), indicating that liquid biopsy method for detecting the pathology of CHB is possible. CONCLUSIONS: This is the first study to systematically elucidate the relationships among gene expressions, clinical parameters and pathological inflammation grades in CHB, and to build models predicting inflammation grades by gene expressions and/or clinical parameters as well.

Increased genome instability in human DNA segments with self-chains: homology-induced structural variations via replicative mechanisms.

Environmental factors including ionizing radiation and chemical agents have been known to be able to induce DNA rearrangements and cause genomic structural variations (SVs); however, the roles of intrinsic characteristics of the human genome, such as regional genome architecture, in SV formation and the potential mechanisms underlying genomic instability remain to be further elucidated. Recently, locus-specific observations showed that 'self-chain' (SC), a group of short low-copy repeats (LCRs) in the human genome, can induce autism-associated SV mutations of the MECP2 and NRXN1 genes. In this study, we conducted a genome-wide analysis to investigate SCs and their potential roles in genomic SV formation. Utilizing a vast amount of human SV data, we observed a significant biased distribution of human germline SV breakpoints to SC regions. Notably, the breakpoint distribution pattern is different between SV types across deletion, duplication, inversion and insertion. Our observations were coincident with a mechanism of SC-induced DNA replicative errors, whereas SC may sporadically be used as substrates of nonallelic homologous recombination (NAHR). This contention was further supported by our consistent findings in somatic SV mutations of cancer genomes, suggesting a general mechanism of SC-induced genome instability in human germ and somatic cells.

Combination of low-intensity pulsed ultrasound and C3H10T1/2 cells promotes bone-defect healing.

AIMS: We systematically investigated the effect of combined use of low-intensity pulsed ultrasound (LIPUS) and bone mesenchymal stem cells C3H10T1/2 on bone-defect healing. METHODS: C3H10T1/2 cells were first induced into a stationary phase by incubation with low fetal bovine serum (5 ml/l) for five days and then sonicated with LIPUS for ten minutes once every day for five consecutive days. The same LIPUS treatment combined with C3H10T1/2 cells, which were incubated in regular fetal bovine serum (10 ml/l) were used to aid femoral fracture healing in Sprague-Dawley rats during four consecutive weeks. C3H10T1/2 cell proliferation activity was detected by MTT assay. Cell-cycle changes were determined, and cell proliferation index was calculated using flow cytometry. Bone reparation was evaluated by X-ray imaging and hematoxylin and eosin (H&E) staining during the healing process. RESULTS: LIPUS promoted C3H10T1/2 cell proliferation, the mechanism of which was possibly the up-regulation of Bmi-1 gene expression. At the end of week two after combined use of LIPUS and C3H10T1/2, the femoral gap was reduced on X-ray images. According to H&E staining results, new bone had homogeneous and similar density compared with normal surrounding bone after combined use of LIPUS and C3H10T1/2. At the end of week four, bone defects could not be observed by X-ray in all four groups and repaired bone substance in all four groups could be observed by H&E staining. CONCLUSIONS: LIPUS treatment effectively promotes C3H10T1/2 cells to enter the growth/split phase from the stationary phase. This process enhances cell proliferation, which consequently promotes bone-defect healing.

Genome-wide CNV analysis in mouse induced pluripotent stem cells reveals dosage effect of pluripotent factors on genome integrity.

BACKGROUND: Induced pluripotent stem cells (iPSCs) derived from somatic cells have enormous potential for clinical applications. Notably, it was recently reported that reprogramming from somatic cells to iPSCs can induce genomic copy number variation (CNV), which is one of the major genetic causes of human diseases. However it was unclear if this genome instability is dependent on reprogramming methods and/or the genetic background of donor cells. Furthermore, genome-wide CNV analysis is technically challenging and CNV data need to be interpreted with care. RESULTS: In order to carefully investigate the possible CNV instability during somatic reprogramming, we performed genome-wide CNV analyses with 41 mouse iPSC lines generated from the same parental donor; therefore, the donor's genetic background can be controlled. Different reprogramming factor combinations and dosages were used for investigating potential method-dependent effects on genome integrity. We detected 63 iPSC CNVs using high-resolution comparative genomic hybridization. Intriguingly, CNV rates were negatively associated with the dosages of classic factor(s). Furthermore, the use of high-performance engineered factors led to less CNVs than the classic factor(s) of the same dosage. CONCLUSION: Our observations suggest that sufficient reprogramming force can protect the genome from CNV instability during the reprogramming process.

The impact of internal-generated contextual clues on EFL vocabulary learning: insights from EEG.

With the popularity of learning vocabulary online among English as a Foreign Language (EFL) learners today, educators and researchers have been considering ways to enhance the effectiveness of this approach. Prior research has underscored the significance of contextual clues in vocabulary acquisition. However, few studies have compared the context provided by instructional materials and that generated by learners themselves. Hence, this present study sought to explore the impact of internal-generated contextual clues in comparison to those provided by instructional materials on EFL learners' online vocabulary acquisition. A total of 26 university students were enrolled and underwent electroencephalography (EEG). Based on a within-subjects design, all participants learned two groups of vocabulary words through a series of video clips under two conditions: one where the contexts were externally provided and the other where participants themselves generated the contexts. In this regard, participants were tasked with either viewing contextual clues presented on the screen or creating their own contextual clues for word comprehension. EEG signals were recorded during the learning process to explore neural activities, and post-tests were conducted to assess learning performance after each vocabulary learning session. Our behavioral results indicated that comprehending words with internal-generated contextual clues resulted in superior learning performance compared to using context provided by instructional materials. Furthermore, EEG data revealed that learners expended greater cognitive resources and mental effort in semantically integrating the meaning of words when they self-created contextual clues, as evidenced by stronger alpha and beta-band oscillations. Moreover, the stronger alpha-band oscillations and lower inter-subject correlation (ISC) among learners suggested that the generative task of creating context enhanced their top-down attentional control mechanisms and selective visual processing when learning vocabulary from videos. These findings underscored the positive effects of internal-generated contextual clues, indicating that instructors should encourage learners to construct their own contexts in online EFL vocabulary instruction rather than providing pre-defined contexts. Future research should aim to explore the limits and conditions of employing these two types of contextual clues in online EFL vocabulary learning. This could be achieved by manipulating the quality and understandability of contexts and considering learners' language proficiency levels.

Somatic nuclear mitochondrial DNA insertions are prevalent in the human brain and accumulate over time in fibroblasts.

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in non-human species 1-3 and recently demonstrated to occur in rare instances from one human generation to the next 4. Here we investigated numtogenesis dynamics in humans in two ways. First, we quantified Numts in 1,187 post-mortem brain and blood samples from different individuals. Compared to circulating immune cells (n=389), post-mitotic brain tissue (n=798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, more brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures WGS design in a human fibroblast model that recapitulates several molecular hallmarks of aging 5. These longitudinal experiments revealed a gradual accumulation of one Numt every ~13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derived SURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7-fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human post-mitotic tissues produce functionally-relevant human Numts over timescales shorter than previously assumed.

Association of polymorphisms in the solute carrier organic anion transporter family member 1B1 gene with essential hypertension in the Uyghur population.

Solute carrier organic anion transporter family member 1B1 (SLCO1B1) is an important hepatic uptake transporter that can transport a wide variety of endogenous compounds, including thyroid hormones and prostaglandin E2. Dysregulation of thyroid hormones and prostaglandin E2 plays a role in the development of hypertension, suggesting that SLCO1B1 might contribute to the aetiology of essential hypertension (EH). In this study, we selected five single nucleotide polymorphisms (SNPs) at the SLCO1B1 gene promoter or coding regions and performed a case-control association study involving 731 unrelated Uyghur subjects, including 374 hypertensive and 357 normotensive individuals, to investigate the potential genetic contribution of SLCO1B1 to the aetiology of EH. Of the five polymorphisms, only one (i.e., rs4149014) showed correlation with EH. The minor allele of SNP rs4149014 at the SLCO1B1 promoter showed association with increased risk for EH (adjusted OR 1.88; 95% CI 1.36-2.60; P= 1.22 × 10(-4)). This study provides preliminary genetic evidence for the role of variant of SLCO1B1 in the susceptibility to human EH in Uyghurs.

Association of a BLVRA common polymorphism with essential hypertension and blood pressure in Kazaks.

Biliverdin reductase A (BLVRA) is a powerful intracellular antioxidant enzyme and an antagonist to insulin-mediated glucose uptake by the cells. Increased oxidative stress and insulin-resistance (IR) are associated with increased risk for hypertension. Therefore, we hypothesized that BLVRA might be attributable to the variation of susceptibility to essential hypertension, and investigated single nucleotide polymorphism (SNP) rs699512 (Thr3Ala), the only common non-synonymous SNP within BLVRA, in population-based samples of 999 Kazak herdsmen from the villages in Xinjiang, China. The minor allele of SNP rs699512 reduced the risk of essential hypertension (age- and gender-adjusted odds ratio 0.76; 95% confidence interval 0.61-0.94; p = 0.010). Single nucleotide polymorphism rs699512 showed association with both systolic and diastolic blood pressures: the minor allele homozygous carriers had lowest systolic and diastolic blood pressures (139.6 mmHg, 89.6mmHg), followed by heterozygous carriers (145.3 mmHg, 92.3 mmHg), and then major allele homozygous carriers (150.3 mmHg, 95.1 mmHg) (p = 0.005 and 0.009, respectively). These findings provide the first genetic evidence for the role of BLVRA on the susceptibility to human essential hypertension and blood pressure.